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Mexico City, September 5th 2017

Lic. Jaime Maussan Flota PRESENTE

Re: Results of old DNA.massive sequencing process.

Dear Lic. Maussan,

I am informing you hereby about the history and status of the ongoing genomic analysis process on tissue samples from two different mummies.

1.- On June 15th, 6 samples from 2 mummies, dated approximately 1500 years ago, from the Nazca region of Peru were received. The samples received are described in Table 1.

2.- On June 24th, the 6 samples DNA was purified. DNA was obtained under sterile conditions, minimizing the risk of contamination, in accordance with the standards recommended for archaeological samples (Shapiro B, Heslington M. 2012). Integrity and total amount of recovered DNA were determined. The DNA had a range of 8,000 base pairs (bp) to 700 bp, obtaining from 8 μg to 0.5 μg..

Table 2 shows the recovered amount of each sample and in Figure 1, the evaluation of the size and amount of the old DNA obtained.

3.- On June 27th, samples 1, 3, 5 and 6 were selected because they had the right DNA amountf to process and were handed over to Dr. Alfredo Mendoza Vargas, Unit Manager Sequencing and identification of the polymorphisms of the National Institute of Genomic Medicine, so that the samples were processed to be read in a massive sequencer (Illumina Myseq). In order to improve the success of sequencing, it has been proposed to repair the DNA of the chemical modifications undergone because of its age. This repair was performed with the New England Biolabs M0309S PreCR® Repair Mix Kit. This one was imported and was used on August 11th.

After the repair and purification, the DNA was quantified and we obtained:

Mummy 1 0.896 ng / ul with approx. 13ulMummy 3 0.132 ng / ul with approx. 13ulMummy 5 0.144 ng / ul with approx. 13ulMummy 6 0.196 ng / ul with approx. 13ul

The realization of massive sequencing was prepared with these DNA libraries. The mummy 1 sample was made with 10 ng and the rest of the samples between 1 and 2 ng with all the volume counted.

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4.- On August 29th, sample sequencing was obtained, reaching approximately 600,000 readings per sample. In the following link, you can find the genomic readings obtained.https://www.dropbox.com/sh/yd9gitxd09ecpux/AAAHtROmZ10yp6u2gEYCsg53a?dl=0

The DNA readings of each sample were evaluated in the Illumina BaseSpace Sequence Hub platform, using an application to align the sequences with the human genome. These results are shown in Figures 2 to 5. In general, it has been found that DNA reads contain about 30% of DNA similar to that of humans. The other sequences are most likely bacterial origin sequences, which is common in this type of samples.

5.- Old DNA readings are currently in a bioinformatic study, to find out what genetic information can be retrieved, to analyze its meaning and to determine genomic sequencing of greater depth is possible to obtain, in order to have more information and to define the ancestral origins of the samples.

While waiting for your comments, I send you my cordial greetings.

Dr. Rogelio A. Alonso Morales.

References: Shapiro B, Heslington M. 2012. Ancient DNA - Methods and Protocols. Humana Press

Table 1.- List of old samples received on June 15th 2017

# ID Legend on the tube Weight in g

1 HAND 00-1 2.38

2 BRAIN 00-10 1.57

3 MARIA B0 HOM 0.56

4 NECK BONE - VERTEBRAE 00-12-VICTORIA 540 MG 0.53

5 HIP BONE02-12 VICTORIA 0.8325 MG 0.33

6 NECK BONE VICTORIA 00-17 PIEL 187 MG 0.17

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Table 2.- Amount of DNA obtained on the old samples.

Sample No Concentration inng/ul

Volume in ul

Total quantity ug

1 110 80 8.80

2 19 60 1.14

3 50 60 3.00

4 11 50 0.50

5 25 85 2.12

6 35 35 1.22

Figure 1.- Evaluation of the DNA quality and uantity. Electroforesis in Agarosa 1%. PM - Marcador molecular weight (DNA lambda / BsteII) .. On the right, list of the molecular weight in pb. At the top, number of each sample. In each track, 5 μl of the

Figures 3-5. - We present the results obtained from old DNA sequences by aligning them on the human genome.

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